MX2014008299A - Single channel mri guidewire. - Google Patents

Abstract

The present application discloses a guidewire for magnetic resonance imaging with a single channel design to reduce complexity and to provide conspicuous tip visibility under MRI. In one embodiment, a guidewire body includes an antenna formed from a rod and a helical coil coupled together. The helical coil can have multiple windings without any gaps between the windings. The rod passes through the windings of the helical coil and is coupled to the helical coil using a conductive joint positioned at an end of the rod and at an end of the helical coil. Insulation can be positioned between the rod and the windings of the helical coil. The configuration allows visibility of the antenna along the length of a rod, except where it enters the windings of the coil. Thus, the tip visibility is enhanced as being separated from the rod.

Description

IDENTIFICATION OF SPECIFIC GENDER OF SPERMATORY CELLS AND EMBRYOS USING BLOCKED NUCLEIC ACIDS FIELD OF THE INVENTION The production of offspring of a predetermined sex, or in a predetermined sex relation, is desirable in a number of industries, including livestock. The separation of specific genus from sperm cells or embryos can facilitate the production of offspring having a predetermined sex. Separate sperm cells can be used in artificial insemination or in vitro fertilization to produce zygotes that evolve in organisms of a predetermined sex. However, techniques for producing populations of sperm cells or embryos that are sufficiently enriched for gender are lacking.

BACKGROUND OF THE INVENTION In one aspect, a method is provided for separating a population of sperm cells or embryos by contacting the population with a labeled blocked nucleic acid capable of binding a specific gender tandem repeat sequence that occurs in a portion of the population. Sperm cells or embryos labeled are then separated from the unlabelled sperm cells.

SUMMARY OF THE INVENTION In one aspect, there is provided a sperm cell or embryo having a specific genus tandem repeat sequence and a labeled oligonucleotide portion, such as a blocked nucleic acid, linked to the specific genus sequence.

In one aspect, a population of sperm cells or embryos having a specific genus tandem repeat sequence occurring on the X or Y chromosome is provided. A portion of the population of sperm cells or embryos have a labeled oligonucleotide portion, the which is a blocked nucleic acid, linked to the specific genre sequence.

In one aspect, the sex of an embryo is identified by contacting at least one cell of the embryo with a blocked nucleic acid. The blocked nucleic acid comprises a tag and has the ability to bind a specific genre tandem repeat sequence present in the cells of either the male or female embryo. Detecting the presence or absence of the label in the embryo makes it easy to identify the sex of the embryo.

In one aspect, a method for directing specific sequence DNA with blocked nucleic acids, such as for use in site-specific modulation of gene expression, or induction of site-specific genomic DNA changes (including mutation, recombination or repair) in living cells.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation showing the location of specific genre tandem repeat sequences (GSTRS) and target sequences on a chromosome.

Figure 2 is a repeated non-expressed sequence of the Y chromosome bobbin showing the location of tandem repeat sequences (GSTRS).

Figure 3 is an illustration showing the structure of a blocked nucleic acid functionalized with pyrene and the functionality of invaded blocked nucleic acids with double-stranded nucleotides. Texp is the experimental temperature and Tm is the dissociation temperature.

Figure 4 is a photograph showing the male bovine somatic nucleus and invasive LNA.

Figure 5 is a photograph showing LNA-Cy3 invader in a repaired male bovine embryo.

Figure 6 is a photograph showing a live Bovine embryo labeled with probe INV-Cy3 and co-labeled with Hoechst 33342 to show the co-localization of INV-Cy3 in the core labeled with Hoechst.

Figure 7 is a photograph showing repaired hybridized porcine sperm for an iLNA probe specific for a Y chromosome sequence.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to the sex identification of sperm cells and embryos and the generation of sperm cell fractions or embryonic fractions that are enriched for the X or Y chromosome., the invention provides methods for separating sperm cells containing a labeled oligonucleotide portion (a blocked nucleic acid) linked to a specific genre tandem repeat sequence or a complement of a specific genre tandem repeat sequence. The oligonucleotide fractions conveniently bound in sufficient number to a region of the chromosome to generate a detectable signal that can be used as a basis to distinguish, and optionally separate cells containing the repeat sequence in tandem of specific gender of those that do not. The invention further provides a method for the separation of sperm cells or embryos carrying an X chromosome from sperm cells or embryos carrying a Y chromosome. The sperm-enriched cell fractions of the genus can be used to fertilize ovules to produce offspring of a predetermined sex . The invention further provides a method for selection of embryos carrying an X chromosome or embryos carrying a Y chromosome. Embryos that have been contacted with labeled blocked nucleic acid are suitably viable, so that the destruction of one or more cells of the embryo can be avoided.

In another aspect the invention relates to the ability to direct specific sequence DNA that can be used for site-specific modulation of gene expression, induction of specific genomic DNA changes (including mutation, recombination or repair) in living cells by binding and specific activation of a blocked nucleic acid.

As used herein, a "specific gender tandem repeat sequence" or "GSTRS" is a non-autosomal chromosome sequence that is repeated on either the Y chromosome or the X chromosome, but not both. Multiple GSTRS occur in a region of the X or Y chromosome, as shown schematically in Figure 1. Figure 2 shows a repeated non-expressed sequence of the bovine Y chromosome showing the location of tandem repeat sequences (GSTRS). GSTRS can occur anywhere on the X or Y chromosome. In some embodiments, the GSTRS targets of the invention occur at or near the end of the chromosome. The specific genre tandem repeat sequence may comprise at least about 10 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 3,000 nucleotides, or at least about 4,000 nucleotides, and less than about 10,000 nucleotides, less than about 9,000 nucleotides, less than about 8,000 nucleotides, less than about 7,000 nucleotides, less than about 6,000 nucleotides, or less than about 5,000 nucleotides. Conveniently there are less than about 50,000 nucleotides, about 10,000 nucleotides, about 5,000 nucleotides, about 3,000 nucleotides, about 2,000 nucleotides, about 1,000 nucleotides, about 500 nucleotides, about 300 nucleotides, about 100 nucleotides, about 100 nucleotides, about 1 nucleotide, or zero nucleotides between each repeated GSTRS unit. The GSTRS does not have to be repeated as exactly the same sequence, and some variation in the repeated sequences is possible without affecting the scope of the invention. The repeated GSTRS units may share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity with each other. Percent identity can be determined using algorithms used in BLASTn or MEGABLAST programs, which can be used to obtain sequences homologous to a reference polynucleotide, as is known in the art. The algorithms used for sequence alignment are described by Tatiana A. Tatusova, Thomas L. Madden (1999), FEMS Microbiol Lett. 174: 247-250. The GSTRS may be repeated at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 750 times, or at least approximately 1000 times on a chromosome.

For each GSTRS, a blocked nucleic acid can be selected to bind to the GSTRS or a complement to the GSTRS. As schematically described in Figure 1, the blocked nucleic acid can direct a shorter white sequence within the GSTRS. As used herein, "white sequence" is a segment of DNA within the GSTRS, wherein the blocked nucleic acid binds the target sequence or the complement of the target sequence. The target sequence may include at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14 nucleotides, at least about 16 nucleotides, or at least about 18 nucleotides. The white sequence may include less than about 100, less than about 90, less than about 80, less than about 70, less than about 50, less than about 40, less than about 30, less than about 20, or less than about 16 nucleotides. The blocked nucleic acid can bind at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 9 nucleotides, at least about 12 nucleotides, at least about 15 nucleotides, nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, or at least about 35 nucleotides of the GSTRS or complement of the GSTRS. The blocked nucleic acid can be attached to less than about 100 nucleotides, less than about 50 nucleotides, less than about 45 nucleotides, less than about 40 nucleotides, or less than about 20 nucleotides of the GSTRS or complement of the GSTRS.

Suitable GSTRS can be selected by searching public databases for DNA sequences that are highly repetitive on only the X or Y chromosome. Suitable target sequences within the GSTRS can be selected by scanning the GSTRS for consecutive purines or consecutive pyrimidines, eg sequences of homopurine or homopyrimidine. The homopurine or homopyrimidine sequences facilitate the joining of oligonucleotide fractions such as blocked nucleic acids to the larger groove of duplex DNA to form a triplex. The white sequence within the specific genre tandem repeat sequence may include, but is not limited to, homopurine or homopyrimidine sequences, as in certain embodiments, blocked nucleic acids have the ability to bind DNA from any sequence, including mixed DNA sequences that include all different nucleotides, and not only homopurines or homopyridamines.

In some modalities the white sequence is itself a repeated unit within the GSTRS. The GSTRS may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 7, at least about 10, at least about 15, at least about 50, less than about 100, or at least about 200 repeating units of white sequence. A GSTRS having a higher number of repeat units will facilitate the joining of more fractions of oligonucleotides to the GSTRS. Suitably, at least about 5, at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 5,000, at least about 25,000, or at least about 50,000 fractions of oligonucleotides bind to the GSTRS.

In some modalities, more than one white sequence can be selected within the GSTRS, or a complement to the GSTRS. A GSTRS that has a higher number of white sequences will facilitate the joining of more fractions of oligonucleotides to the GSTRS. In other embodiments, more than one type of blocked nucleic acid can be selected to bind the GSTRS or a complement of the GSTRS.

The oligonucleotide portion that binds the target sequence is a blocked nucleic acid (LNA), and the invading blocked nucleic acids a (iLNA) are particularly convenient. Blocked nucleic acids (LNA) are modified RNA nucleotides with an extra bridge which "blocks" the ribose in the 3 'endo (North) conformation. The LNA nucleotides can be mixed with DNA or RNA rues. Oligonucleotide fractions are typically chemically synthed. The formation of blocked ribose improves the base stacking and trunk pre-organization. This significantly increases the hybridization (melting temperature) properties of oligonucleotides.

LNA invaders are duplex DNA with "+1 zipper intercatenary arrays" of 2 'amino-alpha-l-LNA monomers functionalized by intercalator. Invasive ANLs facilitate the purpose of unrestricted sequence of double-stranded DNA (DsDNA) under physiologically relevant conditions and have the ability to specifically recognize short mixed white dsDNA sequence.

Current probe technologies, such as TFO (triplex forming oligonucleotides) and ??? (nucleic acids of peptides), typically the target sequence restricts and / or does not require physiological ionic rtance for efficient recognition of dsDNA. LNA invaders, which are duplex probes with dynamic points consisting of +1 monomers N2-piren-functionalized 2 'amino-alpha-L-LNA intercatenary zipper, enable efficient selection of isosequential dsDNAs. ILNA nucleotides increase the strength, sensitivity and specificity of oligonucleotide-based techniques and facilitate sequence-specific selection of dsDNA sequences under physiological conditions.

LNA and iLNA can be chemically synthed. Suitable methods for synthing LNA and iLNA are described in PCT Publication No. WO2011 / 032034, which is incorporated herein by reference in its entirety. Figure 3 illustrates the structure and operation of a suitable invasive LNA. Blocked nucleic acids may show increased binding affinity in the direction of double-stranded DNA (via pairing of Hoggsteen bases), single-stranded DNA (via Watson-Crick base pairing), and single-stranded RNA targets (by mating Watson-Crick bases). Blocked nucleic acids improve discrimination of unequal nucleic acid targets to reduce false positives and effects specific not selected in diagnostic and biological applications. Blocked nucleic acids can also improve stability against degradation by enzymes, such as nucleases.

Formula A functionalized C5 nucleotide convenient for use in a blocked nucleic acid for use in the methods and compositions described herein are shown in formula X. With respect to formula X, R1 can be selected from hydrogen, hydroxyl, thiol, aliphatic, heteroaliphatic, aryl, heteroaryl, charged fractions, and metal complexes. R2 may be selected from hydrogen, aliphatic, heteroaliphatic, aryl, heteroaryl, functional group, protecting groups, a heteroatom-containing compound, such as a phosphorus-containing compound, a nitrogen-containing compound, a compound containing oxygen, a sulfur-containing compound, and a selenium-containing compound. R3 can be selected from hydrogen, a heteroatom-containing compound, such as a phosphorus-containing compound, a nitrogen-containing compound, an oxygen-containing compound, a sulfur-containing compound, and a selenium-containing compound. R4 is a nucleobase selected from natural or non-natural nucleobases. The linking portion may be selected from aliphatic, aryl, heteroaliphatic, and heteroaryl. Y may be selected from oxygen, sulfur, or NR5 wherein R5 is selected from hydrogen, aliphatic, aryl, heteroaliphatic, and heteroaryl; and m + n = 2 to 4.

In certain embodiments, R1 may be selected from ether, carbonyl, nitrile, disulfide, thioether, amine, amino acid, aminoglycosides, carbohydrates, fluorophores, nucleosides, nucleotides, oligonucleotides, peptides, intercalators, lipidoses, sterols, porphyrins, proteins, and vitamins . In particular embodiments, R1 can be selected from amide, ester, carboxylic acid, aldehyde, ketone, spermine derivatives, guanidine groups, spinal labels, electrochemical probes, fatty acids, glycerols, glycols, polyethylene glycol, active redox FRET labels , and ferrocene derivatives. Even very typically, R1 can be selected from hydrogen, hydroxyl, thiol, primary amine, biotin, lauric acid, palmitic acid, stearic acid, fluorescein, rhodamine, cyanine, pyrene, perylene, coronen, adamantine, acridine, phenanthroline, diphenylphosphorylazide, fragment Tat HIV, transport, cholesterol, lithocholic-oleyl, myristoyl, docosanol, lauroil, stearoil, palmitoil, oleoil, and linoleoil, dihydrotestosterone, lithocholic acid, folic acid, and vitamin E.

In certain embodiments the monomer is of the Formula Y Formula Y The blocked nucleic acid that binds to the GSTRS may include a tag that is detectable when bound to the specific genre tandem repeat sequence. Suitable labels include, but are not limited to, inks, fluorescent molecules such as CY3 or CY5, heavy density molecules such as gold or iron, magnetic molecules, nanoparticles, picoparticles, or any combination of them. The labeled blocked nucleic acid binds in sufficient numbers to the GSTRS to produce a detectable signal. The signal may be detectable by any convenient method including, but not limited to, centrifugation, fluorescence, luminescence, microscopy, magnetic force, densitometry, or combinations thereof. Label coupling methods for oligonucleotides are known in the art and can be adapted to be coupled to the blocked nucleic acids described herein.

The blocked nucleic acid binding to the GSTRS may include a reactive chain that is activated when it binds to the specific genre tandem repeat sequence and which may affect, for example, DNA integrity, cell metabolism, viability, motility, fertility, or a combination thereof. Suitable reactive groups include, but are not limited to, amine crosslinkers, toxins, RNA sequences, DNA sequences, enzymes, nanoparticles, picoparticles, or any combination thereof. The activated blocked nucleic acid binds in sufficient numbers to the GSTRS to produce a chain reaction. The chain reaction can affect the cellular integrity of DNA, cell viability, cell motility, metabolism, fertility, and can allow segregation of the selected cell population of the unbound cell population and from there allow the separation, segregation, or discrimination of the cell population, thus affecting the sex ratio after fertilization. Methods of coupling labels to oligonucleotides are known in the art and can be adapted to be coupled to the blocked nucleic acids described herein.

In other embodiments, the blocked nucleic acids can be labeled with labels that are active for fluorescence resonance energy transfer (FRET) or for conditional release activation of specific reactive groups (CRA). Some blocked nucleic acids can be labeled with a FRET or CRA donor, and others can be labeled with a FRET or CRA acceptor. Agitation of the donor label may agitate the acceptor label, and cause the acceptor label to fluoresce or release the activated group. FRET can therefore be used to improve or differentiate the signal of labeled blocked nucleic acids bound to GSTRS intimately in the chromosome and improve the signal-to-noise ratio. Therefore, CRA can be used to affect, inhibit, or modify integrity, metabolism, motility, viability, or fertility. of the selected / activated cell. For example, two blocked nucleic acids can be designated to bind to a target sequence so that the blocked nucleic acids are placed close to each other after binding to the target sequence, eg, a first blocked nucleic acid can be designated to bind base pairs 1 through 12 and a second blocked nucleic acid can be designated to join base pairs 13 through 24 of a blank sequence 24 base pairs in length. When two different blocked nucleic acids are labeled with suitable ink molecules, for example a fluorescent cyano protein (CFP) as a donor and yellow fluorescent protein (YFP) as an acceptor, FRET can be used. Labeled cells can be agitated with light of a convenient wavelength. For example, if they are agitated with a wavelength of 440 nm, CFP will emit light at a wavelength of 480 nm which overlaps with the stirring wavelength of YFP, and will lead to a peak of YPF signal emission at 535 nm when both blocked nucleic acids are together. After activation the process can also release active, toxic groups, RNA, DNA, enzymes, which affect, but are not limited to, life, metabolism, motility and fertility of the selected cell.

In another modality, the label can be suitably a molecule, such as DNA or RNA, or atom bound to the blocked nucleic acid that enhances the activation or deactivation of the physiological process of the cell, and can be toxic and / or facilitate the destruction, inability or inactivation of the cell when joined to a GSTRS. For example, a cellular toxin when bound to the GSTRS may cause the cell to die, may facilitate weakening of cell function, may physiologically disrupt the cell, or may affect cell integrity, so that the cell becomes unviable or incapacitated. The mechanisms by which the tag can affect the cell include, without limitation, an increase in intra-cellular pH, an accumulation of cellular toxins, induction of selective phototoxicity, impairment of mitochondrial function, altered cell motility, acrosomal reaction stimulus. , cell death through a cellular action or the action of electromagnetic waves on the label and combinations thereof. Thus, enriched sperm cell fractions can be generated without the need to separate a viable population of labeled cells from a viable population of unlabeled cells. The labeling can be used in conjunction with, or independently of, one or more detectable labels attached to it or other blocked nucleic acids.

Conveniently, the molecule or atom that facilitates the destruction or incapacity of cellular functions effectively when it is close to other labels, whose labels can be the same or different, and which can each be joined to separate blocked nucleic acids, as could occur after the union of the GSTRS occurs.

Labels can also be used which regulates the capacity, viability, motility, fertility or combination thereof of sperm cells containing a GSTRS. Accordingly, the time in which a labeled sperm cell containing the GSTRS has the ability to fertilize an ovum can be controlled. For example, a sperm cell may be incapacitated in its ability to fertilize an oocyte, inducing premature training, affecting cell motility or motility pattern, or inducing apoptosis or cell death. The fertilization of an ovule can then be delayed for an appropriate amount of time, so that the labeled portion of cells in the population is unable to fertilize the ovule.

Suitable labels which may be used include, for example, noble metals such as silver, gold, platinum, palladium, rhodium and iridium, and alloys and molecules thereof, as well as magnetic compounds, convenient labels may also include siRNA, ions, proteins, peptides, and labels activated after release to affect cell integrity, viability, motility or fertility. Conveniently these labels can be joined as picoparticles or nanoparticles. Cells labeled with the metals or compounds may subsequently be exposed to electromagnetic radiation, such as sono- or radio waves, which may heat and / or agitate the label resulting in the viability of the cell being deteriorated or reduced. Other suitable labels include calcium or calcium containing compounds, ion / calcium activators, pH / hydrogen ion activators, organic compounds with alcohol groups, acids, and denatured enzymes such as trypsin.

The labels may be linked to oligonucleotides using techniques known in the art for coupling molecules in general to oligonucleotides.

In a further embodiment, methods for distinguishing and separating sperm cells or embryos containing a blocked nucleic acid bound to a GSTRS. In some embodiments, sperm cells or embryos are mammals. Conveniently, the sperm cells or Embryos are mammals, fish or birds, or vertebrates. The sperm cells may be of porcine, equine, bovine, ovine, caprine, feline, canine or human origin. In other modalities, sperm cells or embryos are fish or birds. As used herein, a "population" of sperm cells or embryos means at least two sperm cells or at least two embryos. However, the technology can also be used to identify and specifically label an individual embryo (such as for sex determination) or sperm cell (such as to carry out ICSI).

In a first step of the method for identifying genus or for generating embryonic fractions or gendered enriched sperm cells, after washing with buffer and equilibration, the cells are contacted with the labeled oligonucleotide portion. In some embodiments, the cell or cells are permeablilized to facilitate entry of the oligonucleotide into the cells and access to the GSTRS. The cells can be permeabilized by any convenient technique, including but not limited to, osmotic pressure, electroporation, liposomes, impregnated peptides, a modified temperature (eg, increased or decreased) or combinations thereof. In other embodiments, the labeled blocked nucleic acid is passively or actively transported in the cell. The blocked nucleic acid may further include a transport portion, such as a transport peptide, micro or nanoparticles, which facilitates or intervenes in the active absorption of the blocked nucleic acid into the cell. Convenient transport peptides are commercially available from AnaSpec (San Jose, CA, E.U.A.) and include Arg9, TAT, and Cys-TAT. Transport peptides compatible with the ergothionein transporter can also be used.

Once the blocked nucleic acids are bound to the repeated DNA sequence, the sperm cells can be identified and / or separated. Blocked nucleic acid pools labeled in the GSTRS region produce a signal (physical, optical or chemical) that cells containing the GSTRS can be detected and enabled to distinguish themselves from cells that do not contain the GSTRS or not but induce physical reactions , chemical or other that enable the cells contained in the GSTRS but not exclusive, be specifically affected in their integrity, viability, motility, metabolism, fertility or any combination thereof. Once labeled, the cells can be detected or separated, or both detected and separated. Suitable methods for separating cells include, but are not limited to, micromanipulation, centrifugation, magnetic force, flow cytometry, densitometry, or chemical agents that induce changes in metabolism, viability, motility, integrity, fertility or any combination thereof. Suitably, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the cells in the population separated from cells comprise a labeled blocked nucleic acid bound to the GSTRS. The cell population can be separated into a labeled portion containing the GSTRS, and an unlabeled portion that does not contain the GSTRS.

In one embodiment, the tagged portion includes sperm cells containing an X chromosome labeled with the oligonucleotide, and the unlabeled portion includes sperm cells containing Y chromosome not labeled with oligonucleotide. In another embodiment, the tagged portion includes sperm cells that contain a Y chromosome labeled with the oligonucleotide, and the unlabeled portion includes sperm cells that contain an X chromosome not labeled with oligonucleotide. Conveniently, a portion may contain sperm cells in which at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the sperm cells comprise an X chromosome. Alternatively, a portion may contain sperm cells in which at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80 %, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the sperm cells comprise a Y chromosome.

The separated fractions conveniently contain viable sperm cells. As used herein, "viable" refers to a sperm cell that has the ability to fertilize an egg to produce an embryo. Conveniently, a separate sperm portion contains sperm wherein at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80 %, at least about 90%, at least about 95%, or at least about 99% of the sperm cells are viable.

A cellular portion enriched by gender can be Use to fertilize an ovule in vitro or in vivo. Fertilization of an ovule can be achieved, for example, via artificial insemination, including, but not limited to, intra-vaginal, intra-cervical, intra-uterine or surgical insemination, or by intracytoplasmic sperm injection (ICSI). A labeled portion or unlabeled portion can be used for in vivo or in vitro fertilization. The fertilized egg can be allowed to develop to produce an embryo of predetermined sex.

In a further embodiment, the invention provides methods for determining the sex of embryos. Labeled blocked nucleic acids designated to bind to a GSTRS as described above can be incubated with embryos, enter the embryos, and join the GSTRS. As with sperm cells, embryos can be permeabilized to facilitate the entry of blocked nucleic acid labeled to embryos. One or more cells of the embryo may also be removed or biopsied and permeabilized to facilitate entry of the labeled oligonucleotide. The sex of the biopsied cells can then be correlated with the embryo from which the cells were removed. The labeled oligonucleotide portion can also be used to mark and identify a living embryo without affecting the development and fertility of the living embryo. Once that the blocked nucleic acid is bound to the GSTRS, the embryos can be seen under a dissecting microscope or fluorescence microscope to distinguish embryos containing the GSTRS from those that do not contain the GSTRS. As with the sperm cells as described above, the embryo population can be separated into a labeled portion containing the GSTRS, and an unlabeled portion that does not contain the GSTRS.

The following examples are provided to assist in a greater understanding of the invention. The particular materials and conditions employed are intended to be more illustrative of the invention and are not limited to the reasonable scope of the appended claims.

EXAMPLES Example 1: Swine GSTRS, target sequences and corresponding oligonucleotide fractions.

Two complementary (duplex) iLNAs designated "Sequence A" and "Inverse Sequence A" each join the white and reverse-target sequence shown in SEQ ID NO: 2 of the double-stranded DNA. SEQ ID NO: 2 is a 14-nucleotide sequence at nucleotide positions 3231 to 3244 of the GSTRS described in SEQ ID NO: 1. SEQ ID NO: 1 occurs on the porcine Y chromosome and has sequence entry number X12696 (McGraw et al. (1988) Nucleic Acids Research, volume 16, page 10389). Sequence A and Reverse Sequence A were each synthesized with a fluorescent CY3 molecule bound to the 5'-end by means of an ester linkage. The sequences A and inverse A were customized Prof. P. Hrdlicka Biorganic chemistry of the University of Idaho. iLNA were received as a lyophilized powder, and were precipitated in ultrapure water and stored in aliquots at -20 ° C and at -80 ° C.

The DNA sequences repeated in somatic tandem were identified on porcine chromosome 1 with sequence entry number X51555 (SEQ ID NO: 3). It is a DNA sequence 313 base pairs that is repeated approximately 3000 to 6000 times. Two iLNAs designated "Sequence B" and "Reverse Sequence B" were each designated to bind to the blank sequence shown in SEQ ID NO: 4. SEQ ID NO: 4 is a 14-nucleotide sequence at nucleotide positions 120 to 133 of the tandem repeat DNA sequence shown in SEQ ID NO: 3. Both Sequence B and Inverse Sequence B were each synthesized with a fluorescent CY3 molecule linked to the 5'-end by an ester linkage. This somatic DNA sequence (SEQ ID NO: 3) was used as a negative control for experiments.

Examples of porcine GSTRS DNA sequences, target sequences, and oligonucleotide fractions corresponding ones are shown in Table 1 below.

TABLE 1 Example 2: Bovine GSTRS, target sequences, and corresponding oligonucleotide fractions.

A base pair of GSTRS 1399 was identified on bovine X chromosome at locus V00125 (SEQ ID NO: 5) with sequence entry number V00125. Two complementary iLNAs (duplexes) designated "Sequence C" and "Reverse Sequence C" each bind the white and reverse white sequence shown in SEQ ID NO: 5 of double-stranded DNA. SEQ ID NO: 5 is a 20-nucleotide sequence at nucleotide positions 561 to 581 of the GSTRS shown in SEQ ID NO: 5. Sequence C and Reverse Sequence C were each synthesized with a CY3 fluorescent molecule attached to the 5 '. end by means of an ester linkage. The sequences C and inverse C were custom Prof.P. Hrdlicka Biorganic Chemistry from the University of Idaho. iLNA were received as lyophilized powder, and were precipitated in ultrapure water and stored in aliquots at -20 ° C and at -80 ° C.

TABLE 2 Example 3: Method for labeling somatic cells and porcine sperm repaired with CY3-ILL conjugate. Freshly ejaculated porcine semen or defrosted porcine semen (approximately 100 million sperm cells) were added to 10 mL of phosphate buffered saline (PBS). The suspension was centrifuged for 5 minutes at 800 x g. The pellet was precipitated in 1 mL of 3 M NaOH. The suspension was incubated at room temperature for 5 minutes and centrifuged for 5 minutes at 800 x g. The pellet was precipitated in PBS 2 mL and centrifuged for 5 minutes at 800 x g. The pellet was precipitated in PBS or phosphate buffer (PB) to obtain a final concentration of 10 million sperm cells per mL of PBS.

A male somatic nucleus suspension was prepared using standard methods as follows: to. A suspension of male bovine somatic nucleus was prepared using standard methods of: hypotonic treatment of cells using KC1, centrifuging the cells and precipitating them in 3: 1 methanol: acetic acid then storing them (-20 ° C) in this solution until they are ready for its use. b. 0.5 pL of the fixed nucleus were placed on a plastic microscope slide; c. the core was dried and then the slide was heated at 60 ° C for 2 min; d. The labeling buffer was prepared as follows: 1. 500 pL of 10 mM, Tris HCl + lmM EDTA (ie standard TE buffer, pH 7.2) were added to a microcentrifuge tube; 2. 0.5 pL of invading LNA sequence A and reverse sequence A (from a stock solution of 50uM conc in dH20) were added; 3. The mixture was stirred for 2-3 sec. To mix, the solution was maintained at room temperature until needed. and. 300 pL of the labeling buffer was added above the fixed core; F. the slide was placed in a humidification medium at 37 ° C for 3 hr. g. after incubation, the label buffer was washed with the TE buffer and at 37C for 5 min; h. After washing, the slide was dried and then 3.0 μ ?, of mounting medium containing DAPI (ie SlowFade with DAPI, Invitrogen) were added to the sample cover with a coverslip; i. the slide was placed in the microscope stage using a microscope that has fluorescent capabilities; j. The samples were observed in 10X using a DAPI filter to locate the nucleus and then changing to 40X and using the Cy3 filter to stir the Cy3 ink conjugated to the LNA Invader.

After pre-treatment of the sperm cells, labeled LNA duplex CY3 as prepared in Example 1 (designated Sequence A and Reverse A) was incubated with the sperm cells at a final iNNA concentration of 100 ng / mL for 2 hours at 38 ° C. The sperm cells were centrifuged for 5 minutes at 800 x g, the pellet was precipitated in PBST (PBS with 0.05% Tween 20), and the suspension was incubated for 20 minutes at 38 ° C. The sperm cells were centrifuged for 5 minutes at 800 x g, and the pellet was precipitated in PBS or PB. Sperm cells treated with duplex iLNA (Sequence A / Reverse A) labeled CY3 (4μ? _) Were seen under a Zeiss AxioSkop fluorescent microscope. The DAPI chain was optionally added to the sample just before observation with the microscope. Selective binding of labeled CY3 iLNA to the Y chromosome of trimmed swine semen was observed. The swine sperm cells arranged pre-treated with NaOH and RNase A and incubated with chromosome Y specific CY3-ÍLNA Y chromosome stained in red. Somatic porcine chromosomes treated with CY3-ILA of specific Y chromosome were stained red. Somatic porcine chromosomes stained with DAPI that binds DNA and RNA and were stained blue. A fused image of somatic porcine chromosomes stained with DAPI and CY3-ÍLNA was generated. The Y chromosomes appeared to be stained pink, indicating the selective binding of CY3-ILA probe to the Y chromosomes.

It was found that the signals present in 161 of 302 sperm (53.3%) consist of a simple round fluorescent label located centrally on the head of the sperm. The signal was observed as a pleasant point in the nucleus of all male somatic cells.

As a control, freshly ejaculated porcine semen was prepared and permeabilized as described above. A CY3-ÍLNA conjugate with base sequence (CCCTAA) 3, available from the University of Idaho Chemistry Department that binds the telomeres of all mammalian chromosomes were incubated with sperm cells precipitated in PBS at a final iLNA concentration of 00 ng / μ? - for 2 hours at room temperature. Sperm cells (4 μ?,) treated with CY3-ÍLNA (CCCTAA) 3 were seen under a Zeiss AxioSkop fluorescence microscope. Selective binding of CY3-ILA (CCCTAA) 3 was observed to all the porcine chromosome telomeres of the arranged swine semen. Chromosomes stained with 4 ', 6-diamidino-2-phenylindole (DAPI) that does not specifically bind DNA and RNA appeared in blue. In contrast, CY3-iLNA (CCCTAA) 3 stained chromosomes in pink. Figure 4 shows male bovine somatic nucleus labeled similarly labeled with the LNA invader.

Example 4: Labeling method of live bovine sperm cells with CY3-ILL conjugate.

Freshly ejaculated bull semen or thawed bull semen (approximately 100 million sperm cells) were added to 10 mL of phosphate buffered saline (PBS). The suspension was centrifuged for 5 minutes at 800 x g. The pellet was precipitated in PBS or phosphate solution (PB) to obtain a final concentration of 10 million sperm cells per mL of PBS.

After pre-treatment of the sperm cells, labeled LNA duplex CY3 (designated sequence C and inverse C) as prepared in Example 1 was incubated with the sperm cells in a final concentration iNNA of 100 ng / mL for 2 hours at 38 ° C. The sperm cells were centrifuged for 5 minutes at 800 x g, the pellet was precipitated in PBST (PBS with 0.05% Tween 20), and the suspension was incubated for 20 minutes at 38 ° C. The sperm cells treated with duplex iLNA (Sequence C / Reverse C) labeled CY3 (4pL) were seen under a Zeiss AxioSkop fluorescent microscope. The DAPI spot was optionally added to the sample just before observation with the microscope. Selective binding of CY3 iLNA labeled to the Y chromosome of bull semen was observed as red dots in the nucleus. In sperm cells of parallel bull destroyed pre-treated with NaOH and RNase A and incubated with specific chromosome Y CY3-ÍLNA also stained in red. The somatic porcine chromosomes treated with the specific chromosome Y CY3-ÍLNA were also stained with red. Somatic porcine chromosomes stained with DAPI that binds DNA and RNA and were stained blue.

Figure 5 shows the LNA-Cy3 invader in an arranged male bovine embryo.

Example 5: Determination of sex of live bovine embryos Freshly cultured bovine embryos in stage (day 7 ideally) were washed with phosphate saline solution (PBS) and transferred to a well of 40 μL of a microplate (ibidi) that was preloaded with IX PBS pH 7.2 0.5 was added? (100 ng) of sequence iLNA C and inverse C (of an existing concentration of 50 μ in dH20). The microplate containing the embryo was placed in a 37 ° C incubator that was humidified and incubated for 2.5 hr. After incubation the embryos were transferred to another 40 L well containing IX PBS without iLNA. The embryos were washed for 5 min at room temperature, and then the microplate was placed in a microscope stage using a microscope that is provided with fluorescent capacity (Zeiss AxioSkop fluorescent microscope). Embryos are observed at 10X to locate, then viewed at 20X or 40X using fluorescence.

The C iLNA and inverse C duplex probe selects the unique Y-sequence specific chromosome SEQ ID NO: 5. The Y chromosomes were detected as a bright fluorescent red spot within the blastomere nucleus. The absence of signal indicated female embryonic DNA. The accuracy of Sex procedure was demonstrated by means of the determination of the parallel genus of the same embryo using an established PCR method designed for the locus of the male specific gene SRY bovine. Based on 18 bovine embryos produced in vitro generating a result for both trials, there was a 100% coincidence (18/18) of the gender assignment.

Figure 6 shows bovine embryo labeled with probe INV-Cy3 and co-labeled with Hoechst 33342 to show the co-localization of INV-Cy3 in core labeled with Hoechst.

Example 6: In vitro Fertilization of porcine ova with porcine semen enriched with X or Y chromosome Viable sperm cell fractions labeled with CY3-ÍLNA or unlabeled were used to fertilize porcine ova. Approximately 1.5 to 2 hours before preparing the semen, a plate or dish containing 5 to 10 mL of TALP media and a plate or dish containing 5 to 10 mL of FERT media (TALP + caffeine) were prepared and placed in a incubator 38.5 ° C for at least 1.5 hours to balance. Additionally, approximately 30 mL of semen saline solution (0.9% saline + BSA) was placed in a cover to warm to room temperature.

The sperm vision calculation chambers were heated.

To prepare the semen, 2 to 3 mL of the sperm cell portion enriched with chromosome X or Y was increased to 10 mL with saline solution of semen (0.9% saline + BSA). The suspension was centrifuged at 800 x g for 3 minutes. Semen saline solution was reduced to sperm granule, the volume was increased to 10 mL with fresh semen saline solution, the pellet was precipitated in fresh saline, and the suspension was centrifuged. The washing process can be repeated for a total of three times. The final sperm pellet was precipitated in 3 mL of TALP, mixed gently, and a small sample was removed for subsequent sperm motility and concentration determination.

To prepare sperm portion enriched with frozen or thawed X-ray and Y chromosome, a drop of frozen semen (0.5 ce) was placed in a water bath at 50 ° C for 10 seconds. The frozen sperm was then coated on a density gradient and centrifuged at 350 x g for 10 minutes. The pellet was washed once in 2 mL of CellGuard (Minitube, Verona, WI, E.U.A.) and centrifuged at 200 x g for 10 minutes. The pellet was diluted and mixed gently in 1 mL of TALP medium, and a small sample was removed for determination of sperm motility. Sperm motility and concentration was determined using Sperm Vision (Minitube of America, Verona, WI, E.U.A.).

To fertilize the oocytes, 10 L of sperm in FERT media (in a concentration of 2.5 x IO5 sperm / mL) was added to a 500 L well containing 50 oocytes. In vitro fertilization for porcine oocytes is also described in Rath et al., (J. Anim. Sci. 77: 3346-3352 and Long, et al. (1999) Theriogenology 51: 1375-1390), each of which is in the present incorporated as a reference in its entirety.

Example 7: Generation of a labeled iLNA conjugate CY3 and use to identify male and female sperm Imitations of synthetic DNA conjugated to a fluorescent ink were used for in situ detection of Y chromosomes in metaphase preparations of bovine somatic cells and spermatozoa. Using male bovine somatic cells and the Y chromosome as a standard, a synthesis of a conjugated CY3 iLNA was designed and synthesized.

A "C and reverse sequence C" iLNA designated was designed to bind to the target sequence shown in SEQ ID NO: 6. SEQ ID NO: 6 is a 20-nucleotide sequence at nucleotide positions 561 to 581 of the GSTRS shown in the SEQ ID NO: 5. SEQ ID NO: 5 is a bovine Y chromosome sequence up to 60,000 vlces repeated (Perret, J. et al., 1990. A polymorphic satellite sequence maps to the pericentric region of the bovine Y chromosome; Genomics Vol 6 (3) pp 482-490). The iLNA probe designated "Sequence C and inverse C" was synthesized to measure with a fluorescent molecule CY3 linked to the 5 '-end by means of an ester bond: CY3-CAC TAT TAT CGC CAT C The all-sexed sperm generated from flow cytometry was evaluated with iLNA probe (Sequence C) for scoring accuracy. Testing different labeling conditions, it was found that the brief incubation of metaphase chromosomes with iLNA yielded a signal located on the Y chromosome. The sperm population classified by Y showed labeling with the PNA probe on 104 signals on the sperm heads outside of 118 counted. The population classified by X showed labeling with the iLNA specific probe in 8 signals in sperm heads out of 119 counted. In other tests, no signals were present when the female somatic cell chromosomes were incubated with the iLNA probe.

The iLNA signals present in approximately 50% sperm were found to consist of a single round fluorescent spot, centrally located, in the sperm head Unclassified bull sperm provided 23 signals out of 43 sperm heads (53.4%). The iLNA probe was also found to produce signal in male bovine somatic cell lines and in embryos with a similar relationship.

Figure 7 shows porcine sperm hybridized to an iLNA probe specific for a Y chromosome sequence. The Y chromosome resides in the middle of the sperm head.

Example 8: The separation of viable sperm cells fluorescently labeled by flow cytometry The semen will be precipitated in a semen extender (AndroHep CellGuard for porcine sperm, commercially available from initube of America, Verona, I, E.U.A.) to give approximately 1 x 10 7 cells per mL. 1 ng of CY3-ILNA conjugate of Example 1 (Sequence A) will be added to 0.6 mL of sperm cell suspension. The suspension will be incubated at 30C for 2 hours. The absorption of iLNA in the sperm will be verified by means of fluorescent microscopy.

The labeled sperm cells will separate from the unlabeled sperm cells will be separated from the sperm cells without labeling under flow with the following conditions: porcine sperm cells will be separated using a FACSVantage SE with optional DiVa flow cytometer (BD Biosciences, San Jose, CA, E.U.A.) with 100 mW of 488 nm light from a Coherent INNOVO 90C Argon ion laser. A nozzle tip of 100 μm will be used at a shell pressure of 0.08 MPa (12 psi). The cover fluid used will be sterile Dulbecco phosphate saline (DPBS, without Ca2 + or Mg2 +, Sigma-Aldrich, St. Louis, MO, E.U.A.). The detectors used will include FSC-A for advanced dispersion, SSC-A for lateral dispersion, FL1-A with a bandpass filter 530/30 nm to detect any self-fluorescent material, FSC- for double discrimination, and detector FL2-A CY3 with a 585/42 nm band pass filter to detect PNA with fluorescent CY3 tag. A histogram of flow cytometry illustrating the separation of labeled and unlabeled porcine sperm cells will demonstrate the selective binding of CY3-PNA (Sequence A) to the Y chromosome and the separation of sperm with X chromosome from the sperm with Y chromosome. At least 85 % of the cells in the labeled portion are expected to contain the Y chromosome. At least 85% of the cells in the unlabeled portion are expected to contain the X chromosome. This will be validated using PCR from individual sperm cells tested for the presence of the SRY gene.

Example 9: Additional probes for joining bovine and porcine white sequences Tables 3-6 show suitable probes for joining any of the bovine or porcine white sequences shown in Figure 2 (bovine) or SEQ ID NO: 1, which occurs on the porcine Y chromosome and has sequence access number X12696 ( McGraw et al., (1988) Nucleic Acids Research, volume 16, page 10389. Underlined nucleotides show the position of functionalized nucleotides, Cy3 indicates that the probe has been labeled with Cy3, numbers 9 and 4 and N in the sequences shown in Table 6 indicate elements that destabilize the probe Table 3, 4 and 5 also provide the Tm (temperature at which 50% of the probes dissociate from their target or junction dissociation temperature) from the different probes for the target sequences as well as the TA (differential affinity) of the probe against the DNA duplex target.A positive TA indicates higher affinity of the probe for single-stranded DNA.The higher the differential temperature, the stronger the e is the binding to single-stranded DNA.

Table 3 Bovine probes Table 4: Additional Bovine Probes 5 fifteen Table 5: Porcine probes Table 6 Additional Bovine Sequences Sequence 5'-Cv3 AGC CCT GTG 9 CCCTG 3'- TCG GGA CAC? GGG AC Cy3 5 - . 5 -Cy3 AGC CCT GTG 4 CCC TG 3'- TCG GGA CAC 4 GGG AC Cv3 5'-Cv AGC CCT GTG CCC TG 3'- TCG GGA CAC N GGG AC Cv3 5'-Cv3 AGC CCT GTG 9 CCC TG 3 'TCG GGA CAC GGG AC Cv3 5'-Cv¾ AGC CCT GTG CCC TG 3'- TCG GGA CAC 9 GGG AC Cy3 5'-Cv3 AGC CCT GTG 4 CCC TG 3'- TCG GGA CAC GGG AC Cv3 5'-Cv3 AGC CCT GTG CCC TG 3'- TCG GGA CAC 4 GGG AC Cv3 5 'Cy3 AGC CCT GTG CCC TG 3'- TCG GGA CAC GGG AC Cv3 5'-Cv3AGC CCT GTG CCCTG 3'- TCG GGA CAC N GGG AC Cy3 Additional functional modes of probes that can be used for determining gender in animals, most commonly, in cattle, are shown in Table 7, where Cy3 is a Cy3 fluorophore; underlined A / C / G / T are the monomers; and B underlined is a bulky (unpaired) monomer.

Table 7 Destination Region of Probe in Bovine series The following tables 8-10 describe the properties of thermal denaturation of probes that can be used for the determination of the genus of individual cells or multicellular meetings of certain animals and humans; more commonly somatic cells) sperm cells or embryos of certain animals and humans; even more commonly, somatic cells, sperm cells or bovine embryos.

As before, the probes display thermal stabilities ranging from significantly lower to moderately higher than corresponding double-stranded DNA from unmodified targets (note delta Tm values from -13 C to +9, column 4), while white probe duplex (column 2 and 3) are significantly more thermostable (range from +5 to +24 C). Therefore, all probes (which have between two to five +1 zipper monomer arrays) display significantly positive TA values suggesting significant potential for the selection of white double-stranded nucleic acid, most commonly dsDNA.

Table 8: Properties of thermal denaturation of exemplary probes where T = 120Y; A = 120'W; C = 140'X and G = 140'Y Yet another functional example of a particular embodiment is given in Table 9 below, which shows thermal denaturing properties and TA values for modified probes with the unblocked monomer z formula. formula z; R = CH2PY Similar patterns are observed as seen for other described monomers, that is, the probes deploy relatively low thermostability while the white duplex probes are significantly more thermostable. Probes containing one or more +1 zipper arrangement of the unblocked monomer z formula therefore display significantly positive TA values and thus the significant potential for selecting white double-stranded nucleic acid, more commonly white dsDNA.

Table 9 Thermal denaturation properties and TA values for modified probes with the formula z of monomer without blocking Particular embodiments detail the double-stranded probes with certain zipper arrangements of monomers comprising the so-called complementary pseudo-nucleobases (for example, such as 2-thiouracil, 2,6- diamonopurines, inosine and pyrrolo- [2,3-d] -pyrimidine-2- (3H) -one), more commonly, +1 zipper arrangements of monomers comprising pseudo-complementary nucleobases, even more commonly, +1 monomer zipper arrays such as formula Y. Examples of functional examples of these particular embodiments are given in Table 8 below.

Particular additional embodiments detail double-stranded probes with certain zipper arrays (most commonly +1 zippers) of monomers comprising nucleobases wherein, in addition, the opposite nucleotide of the described monomer comprising a pseudo-complementary nucleobase is a described nucleotide or monomer comprising a pseudo nucleobase complementary (eg, said a 2-thiouracil, 2,6-diamonopurines, inosine and pyrrolo- [2,3-d] pyrimidine-2- (3H) -one). For representative functional examples, please see entries 2 and 4 in Table 29 below, where D is a DNA monomer with a nucleobase 2, β-diaminopurine (ie, 2,6-diaminopurine-2'-deoxyriboside) ). With reference to Table 8 below, it was observed that the double-stranded probes with -1 or +1 zipper arrays of the monomeric formula Y display positive TA values, and therefore significant potential for selecting white double-stranded nucleic acid by means of the method described in Figures 1-2, most commonly, ADNds.

With May reference to Table 8 below, it was observed that double-stranded probes with -1 or +1 zipper arrays of the monomeric formula Y, where, in addition, the opposite nucleotide of the monomeric formula Y is D displays positive TA values, and therefore significant potential for select double-stranded double-stranded nucleic acid by means of the method described in figures 1-2, more commonly, Dsds.

Table 10 Double-stranded probes with -1 or +1 arrangements zipper of the monomeric formula It should be understood that the invention is not limited in its application to the details of structure and arrangement of components set out in the above description or illustrated in the drawings. The invention has the capacity of other modalities and of being practiced or of being carried out in several ways. Also, it should be understood that the phraseology and terminology herein used is for the purpose of description and should not be considered as limiting. The use of "including", "comprising", or "having" and variations thereof herein is intended to encompass the concepts listed after and equivalents thereof as well as additional articles. The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention should be considered to cover both the singular and the plural, unless otherwise indicated herein or otherwise. clearly contradicts the context.

The recitation of variations of values in the present are simply intended to serve as a short writing method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated in the specification as if it were individually recited in it. All methods described herein may be carried out in any convenient order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any of the examples, or exemplary language (eg, "such as") provided herein, is intended solely to better illuminate the invention and does not raise a limitation on the scope of the invention unless claimed otherwise. The language in the specification should not be considered as indicating any unclaimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors of carrying out the invention. Variations of those preferred embodiments may become apparent to those skilled in the art upon reading the above description. The inventors expect the experts to employ such variations as appropriate, and the inventors claim that the invention is practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the appended claims to this as permitted by applicable law. Moreover, any combination of the elements described above in all possible variations thereof is encompassed by the invention unless otherwise indicated in the present or is clearly contradicted in another way by context.

Claims (27)

NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following is claimed as property CLAIMS:

1. A method for separating a population of sperm cells or embryos comprising: a) contacting the population with a labeled blocked nucleic acid capable of binding a specific genre tandem repeat sequence in a portion of the population to provide a labeled portion and an unlabeled portion; Y b) separate the labeled portion of the unlabelled portion.

2. The method according to claim 1, characterized in that the blocked nucleic acid comprises an invaded blocked nucleic acid.

3. The method according to claim 1 or 2, characterized in that a plurality of blocked nucleic acids bind to the specific genre tandem repeat sequence in step (a).

4. The method according to any preceding claim, characterized in that the blocked nucleic acid is labeled with a label fluorescent, a heavy density label, a magnetic label, a nanoparticle, a toxin, a DNA, a siRNA, an enzyme, a specific ion, and combinations thereof.

5. The method according to claim 4, characterized in that the population comprises sperm cells and at least 70% of the cells of the tagged portion comprises a Y chromosome and at least 70% of the cells of the unlabeled portion comprise an X chromosome.

6. The method according to claim 4, characterized in that the population comprises sperm cells and at least 70% of cells of the tagged portion comprises an X chromosome and at least 70% of the cells of the unlabeled portion comprise a Y chromosome.

7. The method according to claims 1 to 4, characterized in that the population comprises sperm cells, and wherein at least 50% of the cells of the labeled portion or unlabelled portion are viable after step (b).

8. The method according to any preceding claim, characterized in that the population comprises sperm cells and wherein separating the cells in step (b) comprises physical separation by means of flow cytometry, centrifugation, magnetic force, or chemical separation using processes that affect he metabolism, viability, motility, integrity or fertility.

9. The method according to any preceding claim, characterized in that it further comprises permeabilizing the sperm cells or embryos before or during step (a).

10. The method according to claim 9, characterized in that the sperm cells or embryos are permeabilized using electroporation, liposomes, osmotic pressure, or impregnated peptides.

11. The method according to claim 9, characterized in that it further comprises the use of micro, nano or other particles to facilitate the penetration of the blocked nucleic acid into the sperm cells or embryos before or during step (a).

12. The method according to claim 9, characterized in that the blocked nucleic acid penetrates permeabilized embryos or sperm cells by electroporation, liposomes, nano or microparticles, osmotic pressure, or impregnated peptides.

13. The method according to any preceding claim, characterized in that the specific genre tandem repeat sequence comprises a telomeric sequence.

1 . The method of compliance with any previous claim, characterized in that the specific gender tandem repeat sequence is from about 2,000 to about 10,000 nucleotides.

15. The method according to any preceding claim, characterized in that the labeled oligonucleotide is from about 12 to about 24 nucleotides.

16. The method according to any preceding claim, characterized in that the population comprises mammalian sperm cells or mammalian embryos selected from bovine, porcine, canine, and equine.

17. A sperm cell or embryo comprising a specific gender tandem repeat sequence and a blocked nucleic acid linked to the specific gender tandem repeat sequence.

18. The sperm cell or embryo according to claim 17, characterized in that the blocked nucleic acid is an invaded blocked nucleic acid.

19. The sperm cell or embryo according to claim 17 or 18, characterized in that the blocked nucleic acid is labeled with a fluorescent tag, a heavy density tag, a magnetic tag, a nanoparticle, or combinations thereof.

20. A population of sperm cells, each a cell in the population comprising an X chromosome comprising a specific genre tandem repeat sequence or a Y chromosome comprising a specific genre tandem repeat sequence, wherein at least 30% of the cells comprise a blocked nucleic acid attached to the specific genre sequence.

21. The cells according to claim 20, characterized in that at least 70% of the cells comprise the blocked nucleic acid linked to the specific genus sequence.

22. The cells according to claim 20, characterized in that at least 90% of the cells comprise the blocked nucleic acid linked to the specific genus sequence.

23. A method to identify the sex of an embryo, the method comprising: (a) contacting at least one cell of the embryo with a blocked nucleic acid, the blocked nucleic acid comprising a tag and ability to bind a specific genre tandem repeat sequence, and (b) detect the presence or absence of the label in the embryo.

24. The method according to claim 23, characterized in that at least one cell of the embryo is viable

25. The method according to claim 23 or 24, characterized in that each cell of the embryo is brought into contact with the blocked nucleic acid.

26. The method according to claim 23, 24 or 25, characterized in that the embryo comprises the blocked nucleic acid bound to the specific genre tandem repeat sequence and wherein the embryo is viable.

27. The method according to claim 23, 24, 25 or 26, characterized in that the label comprises CY3 and wherein the label is detected using fluorometric techniques.